Introduction
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered to be the most important insect pest of corn (Zea mays L.) in major corn producing regions of the U.S. (Stamm et al. 1985, Krysan et al. 1986) with crop losses and control costs estimated to be over $2 billion annually in the U.S. alone (Mitchell 2011). WCR larvae are subterranean and specialize on corn roots. WCR larvae will feed on most grasses (Family Poaceae), but can only complete their development on a select few species other than corn (Branson and Ortman 1970, Clark and Hibbard 2004). Larvae are the most economically damaging stage of WCR due to intense feeding on the root system, which can cause major difficulty with nutrient and water uptake in the plants (Kahler et al. 1985, Sutter et al. 1990). This damage can weaken the plant base and cause the plants to fall over or “lodge”, especially during periods of heavy winds and rain, which make harvesting with a combine very difficult.
WCR eggs are laid in soil near the base of the corn plant except where rotation- resistant varieties have evolved that have lost their fidelity to corn and lay their eggs in soybeans and other crops (Onstad et al. 2003, Gray et al. 2009). The larvae use CO2, which is given off by all plants, as a long range attractant as they move through the soil in search of host roots (Branson 1982, Strnad et al. 1986, Hibbard and Bjostad 1988, Bernklau and Bjostad 1998, Miller et al. 2006). Another important volatile is ethylene,
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which is a phytohormone in corn that the larvae use to locate hosts roots (Robert et al. 2012). WCR larvae are also attracted to (E)-β-caryophyllene, which is an induced plant volatile given off when WCR larvae feed on the roots of certain corn varieties. Recently, Robert et al. (2012) discovered that both of these volatiles are used by the larvae to evaluate the health of the plant from a distance. Although older larvae can survive starvation for up to 96 hours, neonate larvae need to locate host roots within 12-36 hours or risk being too weak to burrow into the root (Strnad and Bergman 1987a).
Once the WCR larvae find the roots, contact cues are picked up by the maxillary palps to aid in feeding decisions (Branson and Ortman 1969). Feeding stimulants used by the WCR larvae to identify a host have been identified as a combination of simple
sugars, 30:4:4 mg/ml glucose:fructose:sucrose, and one of the free fatty acids in germinating corn roots, oleic acid or linoleic acid (Bernklau and Bjostad 2008). Interestingly, individual components by themselves did not elicit a major feeding response by the WCR larvae, but together, they did (Bernklau and Bjostad 2008).
Larvae of the WCR have a set of behaviors that help the larvae locate food patches as well as stay within food patches. When WCR larvae are exposed to a substrate that is not recognized as a host and then are removed, they exhibit a “ranging” behavior, where the larvae crawl in a relatively straight direction and move quickly (Strnad and Dunn 1990). Until the larvae encounter host volatiles, they will continue searching in this manner. In contrast, when WCR larvae are exposed to a host root and then are removed, they exhibit a “localized searching” behavior. This behavior involves a restricted area of search with greater number of turns and a decrease in
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speed (Strnad and Dunn 1990, Bell 1991). Throughout their development, WCR larvae move to higher quality, younger root whorls (Apple and Patel 1963, Strnad and Bergman 1987b), and this localized searching behavior likely helps the larvae not stray too far from the root while moving around. These behaviors are important for larval survival and contribute to the highly successful nature of this pest (Strnad and Dunn 1990).
In behavioral bioassays Strnad and Dunn (1990) analyzed the paths that the WCR larvae took after exposure to germinated roots of corn and other grasses. They found that after being exposed to corn and wheat roots, the rootworms initiated localized search. The WCR larvae exposed to giant fox tail and oat (Avena sativa L.) seedling roots, both non-hosts of WCR, showed in part localized search by having a reduced area of search and reduced velocity, however, they did not show any differences in the number of turns and path crossing. Although the rootworm larvae will feed briefly on the oats, they will abandon them due to a feeding deterrent (Branson and Ortman 1969).
Bernklau et al. (2009) found that WCR larvae will initiate localized search when exposed to root extracts, corn root pieces and corn root juice.
Transgenic corn lines with genes from Bacillus thuringiensis (Bt) with resistance to WCR feeding are commonly used for rootworm management in the U.S. These products range from single event hybrids to a pyramided hybrids that have two or more Bt genes targeting rootworms. Current commercially available Bt hybrids targeting the WCR produce one or more of the following proteins mCry3A, Cry3Bb1 or Cry34/35Ab1. SmartStax® is a stacked corn hybrid that is a collaboration between Monsanto Company and Dow AgroSciences, which includes two pyramided rootworm genes, Cry3Bb1 and
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Cry34/35Ab1 as well as three Bt toxins targeted towards above ground pests and herbicides. Syngenta’s next generation product, Agrisure ® Duracade, which includes mCry3A + eCry3.1Ab, is expected to be commercially available in 2014 pending regulatory approval from the USDA. This product has already received FDA and EPA approval.
WCR host recognition behavior is unknown for these transgenic genes and the recent discovery of populations of WCR resistant to Cry3Bb1 Bt corn in the field (Gassmann et al. 2011) raises concerns about the rootworm-transgenic corn
interactions. The objective of this study was to investigate how mCry3A, Cry3Bb1 and Cry34/35Ab1 influence the host recognition behavior of neonate WCR larvae.
Methods
The study was conducted at the USDA-ARS Plant Genetics Research Unit on the University of Missouri-Columbia campus in 2010 and 2011. To assess the host
recognition behavior of WCR neonates on different varieties of corn roots, we
conducted two sets of bioassays. The first set of bioassays consisted of a randomized complete block with nine treatments with WCR larvae susceptible to all Bt corn types on one of seven types of corn, oat (non-host living plant control) or filter paper (control) with 20 replicates per treatment. The seven corn types used included MIR604 (mCry3A), DAS59122-7 (Cry34/35Ab1), MON88017 (Cry3Bb1), SmartStax (Cry3Bb1+Cry34/35Ab1) and their corresponding isolines.
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Insects
WCR eggs were obtained from non-diapausing (Branson 1976) colonies maintained in our laboratory. The egg type used was from an unselected WCR line (Janesville control – see Meihls et al. 2012). WCR eggs were placed in 15 cm × 10 cm oval containers (708 ml, The Glad Products Company, Oakland, CA) and filled
approximately 4 cm deep with a growth medium of 2:1 autoclaved soil and ProMix™ (Premier Horticulture Inc.). The eggs were incubated in the soil at 25°C for approx. two weeks before hatching. Unfed neonate larvae used in the bioassays were used less than 24 hours after hatching.
Plant Material
All of the corn used was soaked in a 10% bleach solution for 10 minutes, rinsed well and allowed to dry completely prior to germination. The corn was then soaked in water at room temperature for 8 hours. After soaking, corn kernels were placed onto a saturated paper towel in closed oval containers and placed in a growth chamber at 25°C to germinate. Oats were treated with a soapy water solution, rinsed well and placed on a saturated paper towel in oval containers for germination in the growth chamber. Upon germination, all plants were kept moist on clean, saturated filter paper in closed oval containers. Corn seedlings were used in bioassays when they reached 3-4 days old; oats were used at 4-5 days old. The roots used in the assays were approximately between 1.5 and 2 inches in length.
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Gene checks were performed on MON88017 and SmartStax roots at the end of the study using QuickStix test strips (EnviroLogix, Portland, ME.
Bioassays
Assays used in this study were modified from Strnad and Dunn (1990). During the bioassays, a single, clean seedling was placed on moistened filter paper in a petri dish and one neonate larva was placed on the root (or on the filter paper for the
control) using a moistened camel’s-hair paintbrush. After exposure to the root for 5 min, the larva was transferred to the center of a specially designed 12.5 cm arena on lightly moistened filter paper and its host-searching behavior was recorded for five minutes using the EthoVision system (Version 3.1, Noldus Information Technology, The
Netherlands). The bioassay was terminated early if any larva exited the arena during the 5 min trial period. No root was reused in the bioassays. Each bioassay resulted in one track file in the EthoVision program.
EthoVision Protocol
The EthoVision arena comprised of a moist 125 mm filter paper circle (Fisher Scientific Pittsburgh, PA), replaced between bioassays, and was placed on a clean glass plate. This was enclosed in a clear acrylic box (20×20×18 cm) mounted under the EthoVision system video camera (Panasonic wv BP334) positioned 0.64 cm above the box with a 15-W fluorescent light located on top for even lighting. For optimum viewing of larvae with the EthoVision system, the tracking settings were set to the following specifications: detection method, subtraction; processing settings, only detect objects
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that are darker than background; scan window of 50 pixels set to search the complete arena; minimum object size, one pixel; maximum object size, 20 pixels; sample rate, 5.994 samples/sec. Recording began after the larvae were placed in the arena and the door to the arena closed. The recording continued for 5 min or until the larva left the filter paper. To account for any changes in the settings due to replacing the filter paper between bioassays, the detection variables were updated before the start of each trial. Parameters measured by the EthoVision system during bioassays included: total distance moved (the distance traveled by the center of gravity of the larva), maximum distance from the origin (the farthest distance traveled by the center of gravity of the larva from the point of origin), mean velocity (cm/s), mean turn angle (the change in direction of movement between two samples), and mean meander (the change in direction of movement of an object relative to the distance it moves). To mitigate image noise and larval body wobbles being recorded as true movement, the following filters and settings were used when calculating the above parameters: total distance moved, downsize filter (1/25) and minimum distance moved (0.2 cm); maximum distance from origin, downsize filter (1/25); mean velocity, downsize filter (1/25); mean turn angle, absolute setting and downsize filter (1/25); mean meander, absolute setting and downsize filter (1/25). Limited larval movement coupled with the above filters
sometimes resulted in no value being calculated for a specific parameter. For trials that did not last the full five minutes as a result of larvae leaving the arena during their search, total distance traveled was adjusted to reflect the distance the larvae would
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have traveled during the five minute period using their average velocity as calculated by the EthoVision software.
Statistical Analysis
An ANOVA was used for these data analyses and was calculated by using the PROC MIXED of the SAS statistical package (SAS Institute 2008). For the mean meander, total distance moved, mean turn angle, maximum distance from origin and the velocity the linear statistical model contained the main plot effect of treatment. Data were transformed by square root (x+0.5) to meet the assumptions of the analysis. Both of the experiments were run as a randomized complete block.
Results
For all parameters that were measured, the two negative controls (moist filter paper and germinated oat seedlings) were significantly different than all corn
treatments (Table 9, Fig. 13). The larvae that were exposed to the controls had
significantly longer paths and traveled farther from the distance from the origin than the larvae exposed to corn plants including the Bt plants (Figs. 13a, b). The larvae exposed to the negative controls traveled significantly faster, turned less and crossed their paths less than the larvae exposed to the corn plants (Figs. 13c,d,e).
Discussion
There were no dramatic differences between the localized search responses of WCR larvae to any of the corn lines tested, however the rootworm larvae consistently
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demonstrated a ranging behavior after contact with the filter paper and oats, indicating that they did not recognize the controls as hosts. This was expected since oats had similar results before (Strnad and Dunn 1990) and may contain a feeding deterrent (Branson and Ortman 1969). Contact cues associated with the roots are the driving factor of host recognition (Branson and Ortman 1969, Strnad and Dunn 1990), and this study demonstrates that each corn type, the stack as well as isoline, contains sufficient contact cues to elicit a localized search response by Cry3Bb1 susceptible larvae when the larvae are removed from the roots. Apparently, the toxins present in the transgenic roots did not turn the plants into non-hosts from the perspective of this assay despite what may have happened in other assays such as Clark et al. (2006).
Higgins et al. (2009) conducted assays that were somewhat similar to the current experiment, except that in their experiment they exposed the insects to artificial diet (modified after Pleau et al. (2002) with and without Cry34/35Ab1 proteins. They
concluded that Cry34/35Ab1 was perceived as a poor host for WCR larvae. However, the factors responsible for host recognition require specific extraction techniques if they are to be separated from corn (Bernklau et al. 2009), and these factors are likely not present in artificial diet. In addition, Cry proteins are tied up in plant cells under normal
circumstances and not directly available to searching larvae as was done by Higgins et al. (2009). In the current studies, all transgenic products were only available to the neonate insect in plants, and all corn lines were recognized as suitable hosts.
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Acknowledgements
Thanks to Anthony Zukoff who helped with all of the bioassays and to the seed companies for providing the seed for these experiments.
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Experiment Analysis df f P
A Distance Moved treatment 4, 149 9.80 <0.0001 Mean Velocity treatment 8, 149 19.16 <0.0001 Mean Turn Angle treatment 8, 147 41.56 <0.0001 Mean Meander treatment 8, 147 36.91 <0.0001 Maximum Distance
from Origin
treatment 8, 147 22.29 <0.0001 B Distance Moved Medium 6,226 56.29 <.0001
Colony 1,226 0.37 0.5411 Medium*Colony 6,226 1.71 0.1195 Mean Velocity Medium 6,233 61.71 <.0001 Colony 1,233 0.07 0.7961 Medium*Colony 6,233 1.99 0.0683 Mean Turn Angle Medium 6,231 277.34 <.0001 Colony 1,231 1.02 0.3134 Medium*Colony 6,231 4.2 0.0005 Mean Meander Medium 6,235 100.85 <.0001 Colony 1,235 0.03 0.8732 Medium*Colony 6,235 2.02 0.0643 Maximum Distance from Origin Medium 6,227 104.4 <.0001 Colony 1,227 3.84 0.0513 Medium*Colony 6,227 3.47 0.0027
Table 9. Effect of treatment on each parameter measured of the movement of the western corn rootworm during Strnad assays from experiment A using susceptible insects and experiment B using both susceptible and resistant insects.
94 0 20 40 60 80 100 120 140 160 180 D is ta n c e t ra v e le d ( m m )
Total Distance Moved
a b b b b b b b a 0 10 20 30 40 50 60 D is ta n c e t ra v e le d ( m m )
Maximum Distance from Origin
a b b b b b b b a 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 V e lo c it y ( m m /s ) Velocity a b b b b b b b a 0 20 40 60 80 100 120 140 M e a n t u rn a n g le ( d e g re e s ) Turn Angle a a a a a a a b b 0 10 20 30 40 50 60 M e a n d e r (d e g re e s /m m ) Meander a a a a a a a b b
Figure 13. The total distance moved, maximum distance from origin, velocity, turn angle and meander of the western corn rootworm larvae in five minutes after exposure to a different plant seedlings or filter paper for experiment A. Letters indicate significant differences between corn types (p≤0.05). Analysis was done with square root transformed data A D E C B
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